Abstract Background Approximately 1 in 25 people admitted to a hospital in the United States will suffer a health care–associated infection (HAI). Environmental contamination of hospital surfaces contributes to HAI transmission. We investigated the impact of an antimicrobial surface coating on HAIs and environmental bioburdens at 2 urban hospitals. Methods A transparent antimicrobial surface coating was applied to patient rooms and common areas in 3 units at each hospital. Longitudinal regression models were used to compare changes in hospital-onset multidrug-resistant organism bloodstream infection (MDRO-BSI) and Clostridium difficile infection (CDI) rates in the 12 months before and after application of the surface coating. Incidence rate ratios (IRRs) were compared for units receiving the surface coating application and for contemporaneous control units. Environmental samples were collected pre- and post-application to identify bacterial colony forming units (CFUs) and the percent of sites positive for select, clinically relevant pathogens. Results Across both hospitals, there was a 36% decline in pooled HAIs (combined MDRO-BSIs and CDIs) in units receiving the surface coating application (IRR, 0.64; 95% confidence interval CI, .44–.91), and no decline in the control units (IRR, 1.20; 95% CI, .92–1.55). Following the surface application, the total bacterial CFUs at Hospitals A and B declined by 79% and 75%, respectively; the percentages of environmental samples positive for clinically relevant pathogens also declined significantly for both hospitals. Conclusions Statistically significant reductions in HAIs and environmental bioburdens occurred in the units receiving the antimicrobial surface coating, suggesting the potential for improved patient outcomes and persistent reductions in environmental contamination. Future studies should assess optimal implementation methods and long-term impacts. A transparent antimicrobial surface coating was applied in 6 units at 2 hospitals. Compared to the 12-month pre-application period, significant declines were noted in hospital-onset infections, the environmental bioburden, and recovery of clinically relevant pathogens during the 12-month post-application period.